牛支原体EF-Tu基因的克隆表达、亚细胞定位及间接ELISA方法建立
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摘要
旨在获得牛支原体延伸因子EF-Tu蛋白,分析其在牛支原体菌体内的分布情况,建立EF-Tu间接ELISA法,为进一步研究牛支原体EF-Tu的生物学功能提供理论依据,也为建立牛支原体有效的血清学诊断方法和亚单位疫苗的研制奠定基础。参照Uniprot数据库中M.bovis PG45株EF-Tu基因序列,应用Overlap PCR扩增获得EF-Tu基因并将其克隆至pMD19-T载体,测序正确后,构建pET32a-EF-Tu原核表达载体,转化大肠杆菌BL21表达菌,IPTG诱导表达,纯化后的表达产物免疫新西兰兔(New Zealand rabbit)制备多克隆抗体,利用间接ELISA测定高免血清抗体效价,用Western blot和间接ELISA法初步定位EF-Tu在菌体内的分布,通过优化反应条件,建立间接ELISA检测法。结果表明:重组蛋白EF-Tu约为66ku,主要以可溶性形式表达;采用间接ELISA测定的多克隆抗体效价为1:6400;Western blot和ELISA表明该蛋白在牛支原体细胞质和细胞膜中均有表达,且含量相当。利用EF-Tu建立的ELISA法具有很好的特异性和敏感性。通过原核表达系统成功得到牛支原体延伸因子的重组蛋白,该蛋白分布于菌体细胞质和膜表面,且含量基本相当。本研究所建立的EF-Tu ELISA法适合大规模的血清学诊断检测。
The purpose of the present study was to establish an indirect enzyme linked immunosorbent assay (ELISA) detection method via obtaining prokaryotic expression products of elongation factor Tu (EF-Tu) referring Mycoplasma bovis (M.bovis) and analyze its subcellular localization in M.bovis,which could provide theoretical basis for studying the biological function of EF-Tu in M.bovis and lays the essential foundation for establishing effective serological diagnosis method and construction of subunit vaccine against M.bovis.In this study,specific primers were designed referred to gene sequence of EF-Tufrom M.bovis PG45 reported in Uniprot.And Overlap PCR-amplified products of EF-Tu of M.bovis was inserted into the pMD19-T vector.With right sequences,prokaryotic expression vector of pET32-EF-Tu was constructed and converted into Escherichia coli (BL21) competent cells.Then,the expression of target protein wasinduced with 1 mmol·L~(-1) of IPTG.After that,the recombinant protein of EF-Tu was purified via Ni-NTA affinity chromatography,with which New Zealand rabbits were immunized to prepare polyclonal antibodies,and the titer of antibody was measured by ELISA.Subsequently,subcellular localization of EF-Tu was analyzed using Western blot and ELISA.The indirect ELISA detection method was established by recombinant protein of EF-Tu.Results were as follows:Recombinant protein of EF-Tu showed an expected molecular mass of 66 kD and was expressed in soluble form.The antibody titer of rabbit serum was 1:6 400.The results of ELISA and Western blot showed that the recombinant protein of EF-Tu showed good immunogenicity,and elongation factor was detected in both cytoplasm and cell membrane of M.bovis with similar expression amounts.The indirect ELISA method for the detection of M.bovis established with EF-Tu showed good specificity and sensitivity.The recombinant protein of EF-Tu was successfully obtained,and elongation factor was expressed in both cytoplasm and cell membrane of M.bovis showing similar expression amounts.The indirect ELISA method showed great potential for serological diagnosis of M.bovis.
The purpose of the present study was to establish an indirect enzyme linked immunosorbent assay (ELISA) detection method via obtaining prokaryotic expression products of elongation factor Tu (EF-Tu) referring Mycoplasma bovis (M.bovis) and analyze its subcellular localization in M.bovis,which could provide theoretical basis for studying the biological function of EF-Tu in M.bovis and lays the essential foundation for establishing effective serological diagnosis method and construction of subunit vaccine against M.bovis.In this study,specific primers were designed referred to gene sequence of EF-Tufrom M.bovis PG45 reported in Uniprot.And Overlap PCR-amplified products of EF-Tu of M.bovis was inserted into the pMD19-T vector.With right sequences,prokaryotic expression vector of pET32-EF-Tu was constructed and converted into Escherichia coli (BL21) competent cells.Then,the expression of target protein wasinduced with 1 mmol·L~(-1) of IPTG.After that,the recombinant protein of EF-Tu was purified via Ni-NTA affinity chromatography,with which New Zealand rabbits were immunized to prepare polyclonal antibodies,and the titer of antibody was measured by ELISA.Subsequently,subcellular localization of EF-Tu was analyzed using Western blot and ELISA.The indirect ELISA detection method was established by recombinant protein of EF-Tu.Results were as follows:Recombinant protein of EF-Tu showed an expected molecular mass of 66 kD and was expressed in soluble form.The antibody titer of rabbit serum was 1:6 400.The results of ELISA and Western blot showed that the recombinant protein of EF-Tu showed good immunogenicity,and elongation factor was detected in both cytoplasm and cell membrane of M.bovis with similar expression amounts.The indirect ELISA method for the detection of M.bovis established with EF-Tu showed good specificity and sensitivity.The recombinant protein of EF-Tu was successfully obtained,and elongation factor was expressed in both cytoplasm and cell membrane of M.bovis showing similar expression amounts.The indirect ELISA method showed great potential for serological diagnosis of M.bovis.
引文
[1]Veterinary Sciences Division of the Agri-Food and Biosciences Institute of Northern Ireland.Disease surveillance in Northern Ireland,October to December2017[J].Vet Rec,2018,182(8):216-220.
[2]FOX L K.Mycoplasma mastitis:causes,transmission,and control[J].Vet Clin North Am:Food Anim Pract,2012,28(2):225-237.
[3]GAGEA M I,BATEMAN K G,SHANAHAN R A,et al.Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves[J].J Vet Diagn Invest,2006,18(1):29-40.
[4]HALE H H,HELMBOLDT C F,PLASTRIDGE WN.Bovine mastitis caused by aMycoplasma species[J].Cornell Vet,1962,52:582-591.
[5]TSCHOPP R,BONNEMAIN P,NICOLET J,et al.Epidemiological study of risk factors for Mycoplasma bovis infections in fattening calves[J].Schweiz Arch Tierheilkd,2001,143(9):461-467.
[6]KAUF A C W,ROSENBUSCH R F,PAAPE M J,et al.Innate immune response to intramammary Mycoplasma bovis infection[J].J Dairy Sci,2007,90(7):3336-3348.
[7]BRAS A L,SULEMAN M,WOODBURY M,et al.A serologic survey of Mycoplasma spp.in farmed bison(Bison bison)herds in western Canada[J].JVet Diagn Invest,2017,29(4):513-521.
[8]STIPKOVITS L,ROSENGARTEN R F.Mycoplasmas of ruminants:pathogenicity,diagnostics,epidemiology and molecular genetics[C].Brussels:European Commission,1999:14-17.
[9]NIANG M,DIALLO M,CISSE O,et al.Pulmonary and serum antibody responses elicited in zebu cattle experimentally infected with Mycoplasma mycoides subsp.mycoides SC by contact exposure[J].Vet Res,2006,37(5):733-744.
[10]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.XIN J Q,LI Y,GUO D,et al.First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(9):661-664.(in Chinese)
[11]季文恒,储岳峰,赵萍,等.牛支原体逃避宿主免疫的研究进展[J].畜牧兽医学报,2017,48(3):393-402.JI W H,CHU Y F,ZHAO P,et al.Advances in the research of immune evasion by Mycoplasma bovis[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):393-402.(in Chinese)
[12]郭雨丝,陈颖钰,赵刚,等.牛支原体病研究进展[J].中国奶牛,2015(14):36-41.GUO Y S,CHEN Y Y,ZHAO G,et al.The progress on Mycoplasma bovis[J].China Dairy Cattle,2015(14):36-41.(in Chinese)
[13]BRKI S,FREY J,PILO P.Virulence,persistence and dissemination of Mycoplasma bovis[J].Vet Microbiol,2015,179(1-2):15-22.
[14]ZIPFEL C,KUNZE G,CHINCHILLA D,et al.Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-Mediated transformation[J].Cell,2006,125(4):749-760.
[15]WIDJAJA M,HARVEY K L,HAGEMANN L,et al.Elongation factor Tu is a multifunctional and processed moonlighting protein[J].Sci Rep,2017,7(1):11227.
[16]MARCOS C M,DE OLIVEIRA H C,DE FTIMADA SILVA J D,et al.Identification and characterisation of elongation factor Tu,a novel protein involved in Paracoccidioides brasiliensis-host interaction[J].FEMS Yeast Res,2016,16(7):fow079.
[17]REGULA J T,UEBERLE B,BOGUTH G,et al.Towards a two-dimensional proteome map of Mycoplasma pneumoniae[J].Electrophoresis,2000,21(17):3765-3780.
[18]KUNERT A,LOSSE J,GRUSZIN C,et al.Immune evasion of the human pathogen Pseudomonas aeruginosa:elongation factor Tuf is a factor H and plasminogen binding protein[J].J Immunol,2007,195(5):2979-2988.
[19]BALASUBRAMANIAN S,KANNAN T R,BASE-MAN J B.The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin[J].Infec Immun,2008,76(7):3116-3123.
[20]BAREL M,HOVANESSIAN A G,MEIBOM K,et al.A novel receptor-ligand pathway for entry of Francisella tularensis in monocyte-like THP-1cells:interaction between surface nucleolin and bacterial elongation factor Tu[J].BMC Microbiol,2008,8:145.
[21]汤宇娇,袁海文,杨美,等.基于TU基因的牛支原体感染病例实验室快速检测[J].贵州畜牧兽医,2017,41(3):1-3.TANG Y J,YUAN H W,YANG M,et al.Rapid detection of mycoplasma bovis case based on TU gene[J].Guizhou Journal of Animal Husbandry&Veterinary Medicine,2017,41(3):1-3.(in Chinese)
[22]张建华.牛支原体流行病学调查及灭活疫苗研制[D].呼和浩特:内蒙古农业大学,2017.ZHANG J H.Epidemiological investigation of mycoplasma bovis and development of inactivated vacciue[D].Hohhot:Inner Mongolia Agricultural University,2017.(in Chinese)
[23]尹正军,岳华,汤承.绵羊肺炎支原体延伸因子Tu基因的分子特性分析[J].畜牧兽医学报,2015,46(2):288-294.YIN Z J,YUE H,TANG C.Molecular characterization of the elongation factor Tu gene in Mycoplasma ovipneumoniae[J].Acta Veterinaria et Zootechnica Sinica,2015,46(2):288-294.(in Chinese)
[24]许健,储岳峰,高鹏程,等.绵羊肺炎支原体免疫蛋白质组学的初步研究[J].中国兽医科学,2012,42(2):150-153.XU J,CHU Y F,GAO P C,et al.Preliminary study on immunoproteomics of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2012,42(2):150-153.(in Chinese)
[25]ZHANG X,CHU Y F,ZHAO P,et al.Characterization of elongation factor Tu of Mycoplasma ovipneumoniae[J].Pak Vet J,2013,33(4):515-519.
[26]蒋菲.绵羊肺炎支原体EF-Tu和HSP70蛋白免疫原性分析及补体ELISA方法的建立[D].北京:中国农业大学,2016.JIANG F.Immunogenicity analysis of Mycoplasma ovipneumoniae EF-Tu,HSP70and development of complement fixition ELISA[D].Beijing:China Agricultural University,2016.(in Chinese)
[27]包世俊,丁小琴,邢小勇,等.滑液支原体WVU1853株免疫相关膜蛋白的初步分析[J].畜牧兽医学报,2017,48(2):316-323.BAO S J,DING X Q,XING X Y,et al.Preliminarily analysis of immune-related membrane proteins from Mycoplasma synoviae WVU1853 strain[J].Acta Veterinaria et Zootechnica Sinica,2017,48(2):316-323.(in Chinese)
[28]BAO S J,GUO X Q,YU S Q,et al.Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J].BMC Vet Res,2014,10:223.
[29]苏炜德,李娜,薛慧文,等.牛支原体pdhα和pdhβ基因的克隆、原核表达及亚细胞的定位[J].农业生物技术学报,2016,24(4):567-575.SU W D,LI N,XUE H W,et al.Cloning and prokaryotic expression of pdhαand pdhβgenes from Mycoplasma bovis and its subcellular Localization[J].Journal of Agricultural Biotechnology,2016,24(4):567-575.(in Chinese)
[2]FOX L K.Mycoplasma mastitis:causes,transmission,and control[J].Vet Clin North Am:Food Anim Pract,2012,28(2):225-237.
[3]GAGEA M I,BATEMAN K G,SHANAHAN R A,et al.Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves[J].J Vet Diagn Invest,2006,18(1):29-40.
[4]HALE H H,HELMBOLDT C F,PLASTRIDGE WN.Bovine mastitis caused by aMycoplasma species[J].Cornell Vet,1962,52:582-591.
[5]TSCHOPP R,BONNEMAIN P,NICOLET J,et al.Epidemiological study of risk factors for Mycoplasma bovis infections in fattening calves[J].Schweiz Arch Tierheilkd,2001,143(9):461-467.
[6]KAUF A C W,ROSENBUSCH R F,PAAPE M J,et al.Innate immune response to intramammary Mycoplasma bovis infection[J].J Dairy Sci,2007,90(7):3336-3348.
[7]BRAS A L,SULEMAN M,WOODBURY M,et al.A serologic survey of Mycoplasma spp.in farmed bison(Bison bison)herds in western Canada[J].JVet Diagn Invest,2017,29(4):513-521.
[8]STIPKOVITS L,ROSENGARTEN R F.Mycoplasmas of ruminants:pathogenicity,diagnostics,epidemiology and molecular genetics[C].Brussels:European Commission,1999:14-17.
[9]NIANG M,DIALLO M,CISSE O,et al.Pulmonary and serum antibody responses elicited in zebu cattle experimentally infected with Mycoplasma mycoides subsp.mycoides SC by contact exposure[J].Vet Res,2006,37(5):733-744.
[10]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.XIN J Q,LI Y,GUO D,et al.First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(9):661-664.(in Chinese)
[11]季文恒,储岳峰,赵萍,等.牛支原体逃避宿主免疫的研究进展[J].畜牧兽医学报,2017,48(3):393-402.JI W H,CHU Y F,ZHAO P,et al.Advances in the research of immune evasion by Mycoplasma bovis[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):393-402.(in Chinese)
[12]郭雨丝,陈颖钰,赵刚,等.牛支原体病研究进展[J].中国奶牛,2015(14):36-41.GUO Y S,CHEN Y Y,ZHAO G,et al.The progress on Mycoplasma bovis[J].China Dairy Cattle,2015(14):36-41.(in Chinese)
[13]BRKI S,FREY J,PILO P.Virulence,persistence and dissemination of Mycoplasma bovis[J].Vet Microbiol,2015,179(1-2):15-22.
[14]ZIPFEL C,KUNZE G,CHINCHILLA D,et al.Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-Mediated transformation[J].Cell,2006,125(4):749-760.
[15]WIDJAJA M,HARVEY K L,HAGEMANN L,et al.Elongation factor Tu is a multifunctional and processed moonlighting protein[J].Sci Rep,2017,7(1):11227.
[16]MARCOS C M,DE OLIVEIRA H C,DE FTIMADA SILVA J D,et al.Identification and characterisation of elongation factor Tu,a novel protein involved in Paracoccidioides brasiliensis-host interaction[J].FEMS Yeast Res,2016,16(7):fow079.
[17]REGULA J T,UEBERLE B,BOGUTH G,et al.Towards a two-dimensional proteome map of Mycoplasma pneumoniae[J].Electrophoresis,2000,21(17):3765-3780.
[18]KUNERT A,LOSSE J,GRUSZIN C,et al.Immune evasion of the human pathogen Pseudomonas aeruginosa:elongation factor Tuf is a factor H and plasminogen binding protein[J].J Immunol,2007,195(5):2979-2988.
[19]BALASUBRAMANIAN S,KANNAN T R,BASE-MAN J B.The surface-exposed carboxyl region of Mycoplasma pneumoniae elongation factor Tu interacts with fibronectin[J].Infec Immun,2008,76(7):3116-3123.
[20]BAREL M,HOVANESSIAN A G,MEIBOM K,et al.A novel receptor-ligand pathway for entry of Francisella tularensis in monocyte-like THP-1cells:interaction between surface nucleolin and bacterial elongation factor Tu[J].BMC Microbiol,2008,8:145.
[21]汤宇娇,袁海文,杨美,等.基于TU基因的牛支原体感染病例实验室快速检测[J].贵州畜牧兽医,2017,41(3):1-3.TANG Y J,YUAN H W,YANG M,et al.Rapid detection of mycoplasma bovis case based on TU gene[J].Guizhou Journal of Animal Husbandry&Veterinary Medicine,2017,41(3):1-3.(in Chinese)
[22]张建华.牛支原体流行病学调查及灭活疫苗研制[D].呼和浩特:内蒙古农业大学,2017.ZHANG J H.Epidemiological investigation of mycoplasma bovis and development of inactivated vacciue[D].Hohhot:Inner Mongolia Agricultural University,2017.(in Chinese)
[23]尹正军,岳华,汤承.绵羊肺炎支原体延伸因子Tu基因的分子特性分析[J].畜牧兽医学报,2015,46(2):288-294.YIN Z J,YUE H,TANG C.Molecular characterization of the elongation factor Tu gene in Mycoplasma ovipneumoniae[J].Acta Veterinaria et Zootechnica Sinica,2015,46(2):288-294.(in Chinese)
[24]许健,储岳峰,高鹏程,等.绵羊肺炎支原体免疫蛋白质组学的初步研究[J].中国兽医科学,2012,42(2):150-153.XU J,CHU Y F,GAO P C,et al.Preliminary study on immunoproteomics of Mycoplasma ovipneumoniae[J].Chinese Veterinary Science,2012,42(2):150-153.(in Chinese)
[25]ZHANG X,CHU Y F,ZHAO P,et al.Characterization of elongation factor Tu of Mycoplasma ovipneumoniae[J].Pak Vet J,2013,33(4):515-519.
[26]蒋菲.绵羊肺炎支原体EF-Tu和HSP70蛋白免疫原性分析及补体ELISA方法的建立[D].北京:中国农业大学,2016.JIANG F.Immunogenicity analysis of Mycoplasma ovipneumoniae EF-Tu,HSP70and development of complement fixition ELISA[D].Beijing:China Agricultural University,2016.(in Chinese)
[27]包世俊,丁小琴,邢小勇,等.滑液支原体WVU1853株免疫相关膜蛋白的初步分析[J].畜牧兽医学报,2017,48(2):316-323.BAO S J,DING X Q,XING X Y,et al.Preliminarily analysis of immune-related membrane proteins from Mycoplasma synoviae WVU1853 strain[J].Acta Veterinaria et Zootechnica Sinica,2017,48(2):316-323.(in Chinese)
[28]BAO S J,GUO X Q,YU S Q,et al.Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein[J].BMC Vet Res,2014,10:223.
[29]苏炜德,李娜,薛慧文,等.牛支原体pdhα和pdhβ基因的克隆、原核表达及亚细胞的定位[J].农业生物技术学报,2016,24(4):567-575.SU W D,LI N,XUE H W,et al.Cloning and prokaryotic expression of pdhαand pdhβgenes from Mycoplasma bovis and its subcellular Localization[J].Journal of Agricultural Biotechnology,2016,24(4):567-575.(in Chinese)